Kiyotaka Yamakawa

Isolation of Clostridium difficile from the Feces and the Antibody in Sera of Young and Elderly Adults

Attempts were made to isolate Clostridium difficile from a total of 431 fecal specimens from 149 young and 213 elderly healthy adults, and 69 elderly adults with cerebrovascular disease but no gastrointestinal disease. C. difficile was isolated from 49 specimens, and the frequency of isolation was 15.4% in healthy young adults, 7.0% in healthy elderly adults, and 15.9% in elderly adults with cerebrovascular disease. Thirty‐four (about 70%) of the 49 C. difficile strains isolated produced cytotoxin which was neutralized by Clostridium sordellii antitoxin in vitro; in both young and elderly adults approximately 30% of the C. difficile isolates were nontoxigenic. The mean concentration of C. difficile in feces was 104.1/g in young adults and 104.6/g in elderly adults, with a range of 102.0 to 106.9/g. Antibody against C. difficile toxin was found in most of the sera obtained from young adults carrying toxigenic C. difficile, but not in sera of elderly adults, no matter how abundant was toxigenic C. difficile in the feces.

A defined growth medium for Clostridium difficile

Minimal requirements of amino acids and vitamins were determined in chemically defined medium for five strains of Clostridium difficile. Cysteine, isoleucine, leucine, proline, tryptophan and valine were essential amino acids for growth of C. difficile. Arginine, glycine, histidine, methionine and threonine enhanced growth. Biotin, pantothenate and pyridoxine were essential vitamins. A defined medium containing the minimal requirements of amino acids and vitamins produced a rapid and heavy growth which was comparable to that in modified brain heart infusion, a complex medium. Adenine was able to substitute for glycine and threonine, suggesting that the two amino acids may be utilized as precursors of purine nucleotides. The defined medium developed here will assist physiological and biochemical studies on C. difficile.

Genetic analysis of type E botulinum toxin-producing Clostridium butyricum strains.

ABSTRACT Type E botulinum toxin (BoNT/E)-producing Clostridium butyricum strains isolated from botulism cases or soil specimens in Italy and China were analyzed by using nucleotide sequencing of thebont/E gene, random amplified polymorphic DNA (RAPD) assay, pulsed-field gel electrophoresis (PFGE), and Southern blot hybridization for the bont/E gene. Nucleotide sequences of the bont/E genes of 11 Chinese isolates and of the Italian strain BL 6340 were determined. The nucleotide sequences of thebont/E genes of 11 C. butyricum isolates from China were identical. The deduced amino acid sequence of BoNT/E from the Chinese isolates showed 95.0 and 96.9% identity with those of BoNT/E from C. butyricum BL 6340 and Clostridium botulinum type E, respectively. The BoNT/E-producing C. butyricum strains were divided into the following three clusters based on the results of RAPD assay, PFGE profiles of genomic DNA digested with SmaI or XhoI, and Southern blot hybridization: strains associated with infant botulism in Italy, strains associated with food-borne botulism in China, and isolates from soil specimens of the Weishan lake area in China. A DNA probe for thebont/E gene hybridized with the nondigested chromosomal DNA of all toxigenic strains tested, indicating chromosomal localization of the bont/E gene in C. butyricum. The present results suggest that BoNT/E-producing C. butyricum is clonally distributed over a vast area.

Enhancement of Clostridium difficile toxin production in biotin-limited conditions

The effect of biotin on toxin production by Clostridium difficile was examined in a defined medium. When toxin production by strain KZ 1647, which was isolated from a healthy adult, was examined in relation to its biotin requirement, it was found that with decreasing concentrations of biotin, bacterial growth was decreased, but production of both toxins A and B were remarkably increased, particularly with 0.05 nM biotin. The time course of production of both toxins in biotin-limited conditions was similar to that in biotin-enriched conditions. The biotin effect on toxin production was also observed in 15 other strains, suggesting that the effect occurs frequently amongst toxigenic C. difficile strains. The biotin effect is discussed in relation to the pathogenesis of C. difficile colitis.

Recovery of spores of Clostridium difficile altered by heat or alkali.

The effect of heating or alkali-treatment on spore recovery in ordinary growth medium was examined for four strains of Clostridium difficile. Heating spores at 80 degrees C for 10 min produced 95.50-99.95% decreases in the recovery rates. Treatment with 0.1 N NaOH for 15 min produced 99.47 and 99.83% decreases in spore recovery rates for two of the four strains. The influence of either addition of lysozyme after treatment with sodium thioglycollate (thioglycollate-lysozyme method) or addition of sodium taurocholate (taurocholate method) on recovery of heat- or alkali-treated C. difficile spores was also examined. Viable spores of all strains altered by heating at 90 degrees C or 100 degrees C for 10 min could not be recovered at all by the taurocholate method. Nor did this method allow recovery of alkali-altered spores treated with greater than 0.2 N NaOH for 15 min. On the other hand, 10-47% of altered spores heated at 90 degrees C for 10 min were recovered by the thioglycollate-lysozyme method, and alkali-altered spores treated with 0.1-0.3 N NaOH for 15 min were as completely recovered by this method as untreated spores. These results indicate that the thioglycollate-lysozyme method is more effective than the taurocholate method for recovery of the heat- or alkali-altered C. difficile spores.

Sporulation and C2 toxin production by Clostridium botulinum type C strains producing no C1 toxin.

All of the 8 strains that were previously assumed to be nontoxigenic Clostridium botulinum type C were re‐examined for their toxigenicity and were demonstrated by trypsinization of the culture filtrates to produce C2 toxin under improved cultural conditions. One per cent glucose added to trypticase peptone medium enhanced C2 toxin production. The larger the spore population, the higher the C2 toxicity and when spore population was smaller than a level of 104/ml, no C2 toxicity was demonstrated. The C2 toxin was produced only during sporulation and not during vegetative growth.

Toxin production by Clostridium difficile in a defined medium with limited amino acids

Basal defined medium (BDM) containing vitamins, minerals and seven amino acids--(/L) tryptophan 0.1 g, methionine 0.2 g, valine 0.3 g, isoleucine 0.3 g, proline 0.3 g, leucine 0.4 g and cysteine 0.5 g--which appeared to be essential for good growth of Clostridium difficile was prepared. Addition of glycine 0.2 g/L and threonine 0.4 g/L to BDM produced better growth of strain VPI 10463, and this defined medium was designated minimum amino acid-defined medium (MADM). Production of toxins A and B by strain VPI 10463 in 6 x MADM containing (/L) tryptophan 0.6 g, methionine 1.2 g, valine 1.8 g, isoleucine 1.8 g, proline 1.8 g, leucine 2.4 g, cysteine 0.5 g, glycine 0.2 g and threonine 0.4 g, was much greater than in MADM. Toxin production by 20 C. difficile strains was examined in two defined media--6 x MADM and complete amino acid-defined medium (CADM) containing 18 amino acids--and one complex medium, modified brain heart infusion medium (m-BHI). Simultaneous production of toxins A and B by all test strains was demonstrated in m-BHI and the two defined media. It was also shown that 6 x MADM was generally better than CADM and as effective as m-BHI for stimulating toxin production by 13 strains. This defined medium would be useful for studies on the physiology, metabolism and pathogenicity of C. difficile.

Cytotoxin production by Clostridium sordellii strains.

A total of 55 strains of Clostridium sordellii, 21 lethal toxin‐positive and 34 lethal toxin‐negative, were tested for cytotoxin production in brain heart infusion medium supplemented with 0.2% Na2HPO4 (m‐BHI) and cooked‐meat‐glucose (CMG) medium using baby hamster kidney (BHK‐21/WI‐2) cells as indicator cells. The m‐BHI medium was preferred to CMG medium and 24 hr of incubation was sufficient for cytotoxin production. Nineteen of the 21 toxigenic strains were also cytotoxigenic, and the strength of the cytotoxigenicity was approximately parallel with that of the lethal toxigenicity. Clostridium difficile antitoxin neutralized C. sordellii cytotoxin and also C. sordellii antitoxin neutralized C. difficile cytotoxin.

Antimicrobial Susceptibility of Clostridium difficile from Different Sources

A total of 79 Clostridium difficile strains from healthy young and elderly adults, elderly patients without gastrointestinal disease, elderly patients receiving antibiotics without gastrointestinal complications, and elderly patients with antibiotic‐associated diarrhea or pseudomembranous colitis were tested for their susceptibilities to 24 antimicrobial agents. All of the 79 strains were inhibited by low concentrations of rifampicin, metronidazole, fusidic acid, vancomycin, ampicillin, and penicillin G. The strains were highly resistant to aminoglycosides, trimethoprim, sulfamethoxazole, nalidixic acid, and cycloserine and often resistant to neomycin, cefoxitin, and cefalexin. Wide variations in the susceptibility of C. difficile strains to erythromycin, clindamycin, lincomycin, chloramphenicol, and tetracycline were found. Strains resistant to erythromycin, clindamycin, and lincomycin were more frequently found among strains isolated from elderly adults than those isolated from young adults, with particularly high frequency among strains isolated from elderly patients receiving antibiotics. None of the 23 strains isolated from healthy young adults was resistant to chloramphenicol. All of the 14 strains resistant to erythromycin, clindamycin, lincomycin, and chloramphenicol were sensitive to tetracycline and all of the 15 strains resistant to erythromycin, clindamycin, lincomycin, and tetracycline were sensitive to chloramphenicol. Only one out of 19 tetracycline‐resistant strains was highly toxigenic, whereas 42 (70%) of 60 sensitive strains were highly toxigenic.

Isolation and characterisation of neurotoxigenic Clostridium butyricum from soil in China

Soil specimens collected from a site around the home of patients with food-borne type E. botulism probably caused by neurotoxigenic Clostridium butyricum in Guanyun, Jiangsu province, China, were examined for the presence of neurotoxigenic C. butyricum. Five lakeside sites of Weishan lake, in an area near to the sites where the type E. botulism outbreaks caused by neurotoxigenic C. butyricum occurred were also surveyed. Type E toxin-producing C. butyricum was isolated from soil from four sites including the site in Guanyun. Polymerase chain reaction assay demonstrated the presence of the type E toxin gene in all the toxigenic isolates. The biochemical properties of the isolates from the Guanyun soil and the lakeside soil were identical except for inulin fermentation and starch hydrolysis properties. These results indicate that neurotoxigenic C. butyricum has its principal habitat in soil.